Light-Enhancing Plasmonic-Nanopore Biosensor for Superior Single-Molecule Detection.

A stacked plasmonic nanowell-nanopore biosensor strongly suppresses the background fluorescence from the bulk and yields net more than tenfold enhancement of the fluorescence intensity. The device offers extremely high signal-to-background (S/B) ratio for single-molecule detection at ultralow excitation laser intensities, while maintaining extremely high temporal bandwidth for single-DNA sensing.

Two color DNA barcode detection in photoluminescence suppressed silicon nitride nanopores.

Optical sensing of solid-state nanopores is a relatively new approach that can enable high-throughput, multicolor readout from a collection of nanopores. It is therefore highly attractive for applications such as nanopore-based DNA sequencing and genotyping using DNA barcodes. However, to date optical readout has been plagued by the need to achieve sufficiently high signal-to-noise ratio (SNR) for single fluorophore sensing, while still maintaining millisecond resolution. One of the main factors degrading the optical SNR in solid-state nanopores is the high photoluminescence (PL) background emanating from the silicon nitride (SiNx) membrane in which pores are commonly fabricated. Focusing on the optical properties of SiNx nanopores we show that the local membrane PL intensity is substantially reduced, and its spectrum is shifted toward shorter wavelengths with increasing e-beam dose. This phenomenon, which is correlated with a marked photocurrent enhancement in these nanopores, is utilized to perform for the first time single molecule fluorescence detection using both green and red laser excitations. Specifically, the reduction in PL and the concurrent measurement of the nanopore photocurrent enhancement allow us to maximize the background suppression and to detect a dual color, five-unit DNA barcode with high SNR levels.

Probing solid-state nanopores with light for the detection of unlabeled analytes.

Nanopore sensing has enabled label-free single-molecule measurements on a wide variety of analytes, including DNA, RNA, and protein complexes. Much progress has been made toward biotechnological applications; however, electrically probing the ion current introduces nonideal noise components. Here we further develop a method to couple an ionic current to a photon-by-photon counting of fluorescent signal from Ca(2+)-sensitive dyes and demonstrate label-free optical detection of biopolymer translocation through solid-state nanopores using TIRF and confocal microscopy. We show that by fine adjustment of the CaCl2 gradient, EGTA concentration, and voltage, the optical signals can be localized to the immediate vicinity of the pore. Consequently, the noise spectral density distribution in the optical signal exhibits a nearly flat distribution throughout the entire frequency range. With the use of high-speed photon counting devices in confocal microscopy and higher photon count rates using stronger light sources, we can improve the signal-to-noise ratio of signal acquisition, while the use of wide-field imaging in TIRF can allow for simultaneous quantitative imaging of large arrays of nanopores.